Synthesis of hydroxamic acid derivatives

ABSTRACT

The present invention describes solid phase reaction products of a solid substrate carrying a plurality of covalently bound hydroxylamine or protected hydroxylamine groups of formula (A) or (B): ##STR1## where P 1  is hydrogen or an amino protecting group, P 2  is hydrogen or a hydroxyl protecting group, and the bond designated (a) covalently links the formula (A) or (B) to the residue of the solid substrate, and is cleavable under acid conditions or by photolysis. The solid phase reaction products can be used for the synthesis of hydroxamic acid derivatives or a combinatorial library of such compounds.

This is a divisional of application Ser. No. 08/809,499, filed Mar. 24,1997, issued as U.S. Pat. no. 5,932,695, which is a § 371 ofPCT/GB96/00428, filed Feb. 26, 1996, which claims priority to GB9503749.5, filed Feb. 24, 1995.

The present invention relates to a solid phase reaction component, andto a process for the use of such a component in the synthesis ofindividual hydroxamic acid derivatives or a combinatorial library ofsuch compounds. In particular, the solid phase reaction component may beused for the synthesis of hydroxamic acids that are inhibitors of zincmetalloproteinase enzymes. Such enzymes are involved in tissuedegradation and the release of tumour necrosis factor from cells.

BACKGROUND TO THE INVENTION

Solid Phase Synthesis

Solid phase synthesis is an established and effective method for thepreparation of peptides, and offers advantages over conventionalsolution phase chemistry in terms of purification and simplicity(Atherton E, Sheppard R C, Solid Phase Peptide Synthesis: A PracticalApproach; IRL Press at Oxford University Press: Oxford, 1989). Solidphase synthesis may also be used for the preparation of non-peptidemolecules (Leznoff C C, Acc. Chem. Res., 1978, 11, 327-333) and recentlythere has been considerable interest in the application of thismethodology to the synthesis of combinatorial libraries for biologicallyactive lead compound optimisation and discovery (Moos W H et al., Annu.Rep. Med. Chem., 1993, 28, 315-324).

Solid phase synthesis requires an appropriate solid substrate whichcarries a plurality of functional groups to which the first reactiveentity in the proposed synthesis may be covalently coupled, and fromwhich the desired molecule may be cleaved after assembly. The solidsubstrate should be compatible with the solvents and reaction conditionsthat are to be used in the peptide or non-peptide synthesis.

The final step in solid phase synthesis is the cleavage of the covalentbond between the desired peptide or non-peptide molecule and the linker.It is desirable that the conditions for the cleavage are orthogonal tothose used during the reactions employed for the synthesis of thepeptide or non-peptide on the solid support such that inadvertentcleavage does not occur during the synthesis. Furthermore, theconditions for cleavage should be relatively mild such that they do notresult in degradation of the desired peptide or non-peptide. Solidsubstrates which present hydroxyl groups as the points of attachment forthe first stage of the synthesis are commonly used, for examplesubstrates which present hydroxyl groups as derivatives of benzylalcohol, the peptide or non-peptide being attached as a benzyl ester andcleaved by hydrolysis, transesterification or aminolysis to release thepeptide or non-peptide as a carboxylic acid, carboxylate ester or as acarboxamide. Also used are substrates which present amino groups, forexample as derivatives of diphenyimethylamine, the peptide ornon-peptide being attached as a carboxamide and cleaved by hydrolysis torelease the peptide or non-peptide as a carboxamide. Substitution ofsuch linkers by a nitro group can enable the photolytic cleavage of thepeptides or non-peptides from the residue of the solid substrate.

Hydroxamic Acid Derivatives

Certain hydroxamic acid derivatives possess useful biologicalactivities. Examples of such hydroxamic acids include compounds thatinhibit urease (Odake S et al., Chem. Pharm. Bull., 1992, 40,2764-2768), trypanosome glycerol-3-phosphate oxidase (Grady et al.. MolBiochem. Parasitol, 1986, 19, 231-240), dehydropeptidase-1(EP-B-276,947), ribonucleotide reductase (Farr R A et al., J. Med.Chem., 1989, 32, 1879-1885), 5-lipoxygenase (Kerdesky F A J et al.,Tetrahedron Lett., 1985, 26, 2134-2146; U.S. Pat. No. 4,731,382),substance P degradation (Laufer R et al, Eur. J. Biochem., 1985, 150,135-140), cardiovascular metalloproteinase enzymes (Turbanti L et al.,J. Med. Chem., 1993, 36, 699-707; WO-9428012) and matrixmetalloproteinase enzymes (Schwartz M A, Van Wart H E, Prog. Med. Chem.,1992, 29, 271-334).

Compounds which have the property of inhibiting the action ofmetalloproteinases involved in connective tissue breakdown such ascollagenase, stromelysin and gelatinase (known as "matrixmetalloproteinases", and herein referred to as MMPs) are thought to bepotentially useful for the treatment or prophylaxis of conditionsinvolving such tissue breakdown, for example rheumatoid arthritis,osteoarthritis, osteopenias such as osteoporosis, periodontitis,gingivitis, corneal, epiderrmal or gastric ulceration, and tumourmetastasis, invasion and growth. It has been found that hydroxamic acidMMP inhibitors can also inhibit the production of the cytokine tumournecrosis factor (herein referred to as "TNF") (Mohler et al., Nature,1994, 370, 218-220; Gearing A J H et al., Nature 1994, 370, 555-557;McGeehan G M et al., Nature 1994, 370, 558-561). Compounds which inhibitthe production or action of TNF are thought to be potentially useful forthe treatment or prophylaxis of many inflammatory, infectious,immunological or malignant diseases. These include, but are notrestricted to, septic shock, haemodynamic shock and sepsis syndrome,post ischaemic reperfusion injury, malaria, Crohn's disease,mycobacterial infection, meningitis, psoriasis, congestive heartfailure, fibrotic disease, cachexia, graft rejection, cancer, autoimmunedisease, rheumatoid arthritis, multiple sclerosis, radiation damage,toxicity following administration of immunosuppressive monoclonalantibodies such as OKT3 or CAMPATH-1 and hyperoxic alveolar injury.Since excessive TNF production has been noted in several diseases orconditions also characterised by MMP-mediated tissue degradation,compounds which inhibit both MMPs and TNF production may have particularadvantages in the treatment or prophylaxis of diseases or conditions inwhich both mechanisms are involved.

Classes of MMP Inhibitors

The known hydroxamic acid MMP inhibitors may be grouped into threeclasses; i) peptidyl hydroxamates, ii) succinyl hydroxamates and iii)arylsulfonamido hydroxamates.

i) The following patent publication discloses peptidyl hydroxamicacid-based MMP inhibitors:

EP-A-345359 (Fuji)

The tri- and tetra-peptidyl hydroxamic acid derivatives disclosed in theabove publication and described elsewhere (Odake S et al., Chem. Pharm.Bull., 1990, 38, 1007-1011; Odake S et al., Chem. Pharm. Bull., 1991,39, 1489-1494; Odake S et al., Biochem. Biophys. Res. Commun., 1994,199,1442-1446) can be regarded as having the following basic structure (I):

    R.sub.1 --X.sub.1 --X.sub.2 --X.sub.3 --NHOH               (I)

wherein the four groups R₁, X₁, X₂ and X₃ may vary according to thedetailed disclosure of each of the publications.

ii) The following patent publications disclose succinyl hydroxamicacid-based MMP inhibitors:

U.S. Pat. No. 4,599,361 (Searle)

EP-A-0236872 (Roche)

EP-A-0274453 (Bellon)

WO 90/05716 (British Bio-technology)

WO 90/05719 (British Bio-technology)

WO 91/02716 (British Bio-technology)

EP-A-0489577 (Celltech)

EP-A-0489579 (Celitech)

EP-A-0497192 (Roche)

WO 92/13831 (British Bio-technology)

WO 92/22523 (Research Corporation Technologies)

WO 93/09090 (Yamanouchi)

WO 93/09097 (Sankyo)

WO 93/20047 (British Bio-technology)

WO 93/24449 (Celltech)

WO 93/24475 (Celltech)

U.S. Pat. No. 5,256,657 (Sterling Winthrop)

EP-A-0574758 (Roche)

WO 94/02446 (British Bio-technology)

WO 94/02447 (British Bio-technology)

WO 94/21612 (Otsuka)

WO 94/25434 (Celltech)

WO 94/25435 (Celltech)

The hydroxamic acid derivatives disclosed in the above publications canbe regarded as having the following basic structure (II): ##STR2##wherein the five substituents R₂ -R₆ may vary according to the detaileddisclosure of each publication. The balance of intrinsic level ofactivity, degree of specificity of inhibition of particular categoriesof MMP, physicochemical and pharmacokinetic properties can vary in anunpredictable way as the substituents R₂ -R₆ are varied.

iii) The following patent publication discloses arylsulfonamidohydroxamic acid-based MMP inhibitors:

EP-A-606046 (Ciba-Geigy)

The hydroxamic acid derivatives disclosed in the above publication canbe regarded as having the following basic structure (III): ##STR3##wherein the four substituents R₇ -R₉ and Ar may vary according to thedetailed disclosure of the publication.

BRIEF DESCRIPTION OF THE INVENTION

A key step in the synthesis of many hydroxamic acid derivatives is thereaction of a carboxylic acid group with hydroxylamine or a protectedhydroxylamine by first activating the carboxylic acid or by conductingthe reaction in the presence of an activating agent. In a recent patentapplication the possibility that peptidyl hydroxamic acids might beprepared using solid phase synthesis was recognised (WO 94/28012).However, the proposed protocol involved first synthesis of the peptideon a solid support followed by cleavage from the support and subsequentconversion of the C-terminal carboxylic acid group to a hydroxamic acidby a method analogous to that indicated above.

It was the hypothesis of the present inventors that hydroxamic acidderivatives might be prepared by the reaction of a solid substratepresenting hydroxylamine groups as the point of attachment for the firstreactive entity of the synthesis, then reacting the resultant solidphase intermediate with the further reactive entities required for thedesired synthesis, followed by a cleavage step to release the finalhydroxamic acid derivative. Such a hydroxylamine-presenting solid phasereaction component could simplify the synthesis and purification ofbiologically active hydroxamic acid derivatives, for example MMPinhibitors of the three classes referred to above, and could beapplicable to the automation of such syntheses. In addition it wouldenable the solid phase synthesis of combinatorial libraries ofhydroxamic acids. Thus, this invention makes available suchhydroxylamine-presenting solid phase reaction components, and provides aprocess for their use in solid phase synthesis of hydroxamic acidderivatives.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, in one of its aspects, a solid phasereaction component comprising a solid substrate, substantially insolublein aqueous or organic reaction media, carrying a plurality of covalentlybound hydroxylamine or protected hydroxylamine groups of formula (A) or(B) ##STR4## wherein P₁ is hydrogen or an amino protecting group and P₂is hydrogen or a hydroxyl protecting group, and the bond designated (a)is one which covalently links the group (A) or (B) to the residue of thesolid substrate and is cleavable under acid conditions or by photolysis.

The bond designated (a) covalently links the group (A) or (B) to theresidue of the solid substrate. The residue of the solid substrate willusually comprise a base substrate carrying suitable linker groups whichindirectly link the hydroxylamine group (A) or (B) to the basesubstrate. Suitable base substrates include those known in the art ofsolid phase peptide synthesis (see for example those described inStewart J M and Young J D, Solid Phase Peptide Synthesis, 2nd Ed; PierceChemical Company: Rockford, Ill., 1984). They include inorganicsubstrates, for example kieselguhr, silica gel and controlled poreglass, and polymeric organic substrates, for example polystyrene,polypropylene, polyethylene glycol, polyacrylamide, cellulose, as wellas composite inorganic/polymeric substrates such as polyacrylamidesupported within a matrix of kieseiguhr particles. Such known basesubstrates include amino and hydroxy functionalised solid substrates. iethose which are chemically modified by introduction of amino or hydroxylgroups, to serve as convenient points for further chemical manipulation.Examples of particular amino or hydroxy functionalised base substratesare: hydroxymethyl copoly(styrene-1 or 2% divinylbenzene), which can berepresented as ##STR5## wherein P represents the polymer backbone;

benzhydrylamine copoly(styrene-1 or 2% divinylbenzene) ("BHA Resin") ormethyl benzhydrylamine copoly(styrene-1 or 2% divinylbenzene) ("MBHAResin"), which can be represented as ##STR6## wherein P represents thepolymer backbone and R is hydrogen or methyl respectively;

polyethylene glycol polystyrene ("PEG-PS");

poly(dimethylacrylamide)polystyrene composite ("Polyhipe");

polyacrylamide Kieselguhr composite ("Macrosorb"); or functionalisedcontrolled pore glass.

Thus, in the case of peptide synthesis, hydroxyl- or amino-carryinglinker groups can be introduced onto such amino and hydroxyfunctionalised base substrates. In case of a hydroxyl-carrying linkergroup, the first amino acid of the peptide to be constructed may beattached as an ester formed between the linker-presented hydroxyl groupand the carboxyl group of the amino acid. In the case of amino-carryinglinker groups, the first amino acid of the peptide to be constructed maybe attached as a carboxamide formed between the linker-presented aminogroup and the carboxyl group of the amino acid.

In an analogous fashion, the solid phase reaction components of thepresent invention may comprise a base substrate, for example of the kindreferred to above, and a linker group which presents the hydroxylaminegroup (A) or (B) for reaction with the first reactive entity in theproposed synthesis. A preferred embodiment of this aspect of theinvention is a solid phase reaction component, comprising a solidsubstrate, substantially insoluble in aqueous or organic reaction media,carrying a plurality of groups of formula (IV): ##STR7## wherein

R₁₀ represents hydrogen or (phenyl)CH₂ --, optionally substituted in thephenyl ring by one or more substituents selected from C₁ -C₆ alkyl, C₁-C₆ alkoxy, halogen, nitrile or NO₂ ;

R₁₁ represents hydrogen, C₁ -C₆ alkyl, or phenyl optionally substitutedby one or more substituents selected from C₁ -C₆ alkyl, C₁ -C₆ alkoxy,halogen, nitrile or NO₂ ;

R₁₂ and R_(12A) independently represent hydrogen, C₁ -C₆ alkyl, C₁ -C₆alkoxy, halogen, nitrile or NO₂ ;

R₁₃ represents a group --(X¹)_(q) --Y-- wherein q is 0 or 1, X¹represents --C(═O)--,

--CH₂ --, --CH₂ C(═O)--, --O(CH₂)_(n) C(═O)--, --O(CH₂)_(n)C(═O)--(A¹)_(m) --, or

--O(CH₂)_(n) C(═O)--(A¹)_(m) --B¹ --, wherein n is an integer from 1 to6, m is 0 or 1, A¹ represents --O--CH(R¹)--NH-- wherein R¹ is the sidechain of a natural or unnatural alpha amino acid, B¹ represents a spacergroup --NH(CH₂)_(p) -- wherein p is 0 or an integer from 1 to 6, and Yrepresents --O-- or --NH--.

In this embodiment, the group (IV) is linked to the solid substrate via"Y", defined as --O-- or --NH--. It will be apparent that these "Y"groups may be incorporated during synthesis of the solid phase reactioncomponent of the invention, by starting with an amino or hydroxyfunctionalised base substrate, for example hydroxymethylpolystyrene,hydroxymethyl copoly(styrene-1% divinylbenzene),benzhydrylaminepolystyrene, benzhydrylamine copoly(styrene-1%divinylbenzene ("BHA Resin"), methyl benzhydrylaminepolystyrene, methylbenzhydrylamine copoly(styrene-1% divinylbenzene, ("MBHA Resin");polyethylene glycol polystyrene ("PEG-PS");poly(dimethylacrylamide)polystyrene composite ("Polyhipe");polyacrylamide Kieselguhr composite ("Macrosorb"); or functionalisedcontrolled pore glass.

Another preferred embodiment of this aspect of the invention is a solidphase reaction component, comprising a solid substrate, substantiallyinsoluble in aqueous or organic reaction media, carrying a plurality ofgroups of formula (IVA): ##STR8## wherein R₁₀ R₁₁ R₁₂ and R_(12A) are asdefined in formula (IV), R_(11A) is as defined for R₁₁ in formula (IV),and R_(13A) is a bond or is as defined for R₁₃ in formula (IV).

As used herein the term "C₁ -C₆ alkyl" means a straight or branchedchain alkyl moiety having from 1 to 6 carbon atoms, including forexample, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, t-butyl, pentyl and hexyl. "C₁ -C₆ alkoxy" means an alkoxygroup wherein the alkyl part is C₁ -C₅ alkyl.

In the preferred solid phase reaction component of the inventioncarrying a plurality of groups of formula (IV), referred to above:

R₁₀ may for example be hydrogen, 4-methoxybenzyl or 2,4-dimethoxybenzyl.Presently preferred are compounds in which R₁₀ is hydrogen.

R₁₁ may for example be hydrogen, methyl, phenyl, 4-methylphenyl,4-methoxyphenyl or 2,4-dimethoxyphenyl. Presently preferred arecompounds in which R₁₁ is hydrogen.

R₁₂ may for example be hydrogen or methoxy. Presently preferred arecompounds in which R₁₂ is hydrogen.

R₁₃ together with the base substrate to which it is attached may forexample be oxymethyl-copoly(styrene-1 divinylbenzene)resin,oxymethylcopoly(styrene-2% divinylbenzene)resin,oxyacetomidomethyipolyethyleneglycol-copoly(styrene-1%divinylbenzene)resin oroxyacetomidomethylpolyethyleneglycol-copoly(styrene-2%divinylbenzene)resin. Presently preferred are compounds in which R₁₃together with the base substrate to which it is attached isoxymethyl-copoly(styrene-1% divinylbenzene)resin.

Specific preferred solid phase reaction components of the inventioncarrying a plurality of groups of formula (IV), are:

4-(O-Methylhydroxylamine)phenoxymethyl-copoly(styrene-1%divinylbenzene)-resin (100-200 mesh),

4-[4-(O-Methylhydroxylamine)-3-methoxyphenoxy]-(N-4-methylbenzhydryl)butyramide-copoly(styrene-1%-divinylbenzene)-resin(100-200 mesh),

4-(2',4'-Dimethoxyphenyl-O-methylhydroxylamine)-phenoxymethyl-copoly(styrene-1%-divinylbenzene)-resin(100-200 mesh), and

4-[4-(1-Aminooxyethyl)-2-methoxy-5-nitrophenoxy]-(N-4-methylbenzhydryl)butyramide-copoly(styrene-1%-divinylbenzene)-resin(100-200 mesh).

A specific preferred solid phase reaction component of the inventioncarrying a plurality of groups of formula (IVA), is:O-Hydroxylamine-2'-chlorotritylcopoly(styrene-1%-divinylbenzene)-resin(200-400 mesh)

Solid phase reaction components of the invention may be prepared bystandard synthetic techniques from solid substrates which are in generalcommercially available or readily derivable from commercially availablematerials. For example, the preferred solid phase reaction component ofthe invention carrying a plurality of groups of formula (IV), referredto above may be prepared by treating a solid substrate, substantiallyinsoluble in aqueous or organic reaction media, carrying a plurality ofbenzyl alcohol groups of general formula (V) ##STR9## wherein R₁₁ -R₁₃are as defined for general formula (IV) with a compound of formula (VI)

    R.sub.10 NHOH                                              (VI)

wherein R₁₀ is as defined for general formula (IV) but is other thanhydrogen under Mitsunobu reaction conditions using triphenylphosphineand diethylazo-dicarboxylate or similar reagents. The preferred solidphase reaction component of the invention carrying a plurality of groupsof formula (IV), referred to above, wherein R₁₀ is hydrogen may beprepared by treating a solid substrate, substantially insoluble inaqueous or organic reaction media, carrying a plurality of groups ofgeneral formula (VII) ##STR10## wherein R₁₁ -R₁₃ are as defined forgeneral formula (IV) with hydrazine.

A solid substrate, substantially insoluble in aqueous or organicreaction media, carrying a plurality of groups of general formula (VII)can be prepared by coupling a phthalimide derivative of formula (VIII)with a solid substrate, substantially insoluble in aqueous or organicreaction media, carrying a plurality of benzyl alcohol groups of generalformula (V) defined above ##STR11## wherein R₁₁ -R₁₃ are as defined ingeneral formula (IV) under Mitsunobu reaction conditions usingtriphenylphosphine and diethyl azodicarboxylate or similar reagents.Solid substrates, substantially insoluble in aqueous or organic reactionmedia, carrying a plurality of groups of general formula (V), andcompounds of formulae (VI) and (VIII), are known in the art or may beprepared by procedures known to those skilled in the art.

In an analogous manner, the preferred solid phase reaction components offormula (IVA) above may be prepared. Alternativley, the hydroxylaminegroup may be introduced by displacement of a leaving group present inthe base substrate, eg a halogen group such as chloro, using a suitablyprotected hydroxylamine derivative, eg a compound of formula (Viii)above.

The choice of solvent for syntheses based on a solid phase reactioncomponent of the invention will of course depend on the nature of thereagents to be reacted with such component, but will also be influencedby the nature of that component. For example many of the polymerreaction components of the invention may swell to a greater or lesserextent in certain solvents, and generally such swelling will bedesirable for the efficience of the reaction with other reagents in thedesired synthesis.

Solid phase reaction components of the invention presenting groups oftype (B) abovewill generally be accessable (i) by displacement of aleaving group (eg a triflate, mesylate or halogen group) from thedesired base substrate carrying such leaving groups, using hydroxylamineor O-protected hydroxylamine, or (ii) by reacting the desired basesubstrate carrying carbonyl groups with hydroxylamine or O-protectedhydroxylamine to form oxime groups, and then reducing the oxime doublebond, eg using a metal hydride.

In another of its aspects, the present invention comprises a process forthe preparation of a desired synthetic product whose structure ischaracterised by the presence of a covalently bonded hydroxamic acidgroup --CONHOH, which process comprises the steps of:

(i) forming a mixture of a liquid reaction medium and a solid phasereaction component which is substantially insoluble in the said liquidreaction medium and carries a plurality of covalently bound moieties offormula (A1) or (B1) ##STR12## in which formulae X represents theresidual, non-hydroxamate, partial structure of the desired product, P₁represents hydrogen or an amino-protecting group, P₂ represents hydrogenor a hydroxyl protecting group, and the bond designated (a) is one whichcovalently links the moieties (A1) or (B1) to the residue of the solidsubstrate and is cleavable under acid conditions or by photolysis; and

(ii) in the resultant mixture, cleaving the said bond designated (a)and, if P₁ or P₂ as the case may be is not hydrogen, removing thatprotecting group P₁ or P₂ before, after or during cleavage of bond (a);and

(iii) separating the resultant liquid reaction phase from the resultantreaction solids and recovering the desired product from the separatedliquid reaction phase.

Trifluoroacetic acid will generally be suitable for the acid hydrolysisof the bond (a). Depending on the structure of the linker group,solutions of trifluoroacetic acid ranging from 95% v/v to 1% v/v may beused. In Example 5 below, the linker group is photolytically cleavable,and in the remaining Examples it is cleavable by acid hydrolysis.

In step (i) of the above process the solid phase reaction componentwhich carries a plurality of moieties of formula (A1) or (B1) may bederived by appropriate chemical modification from the solid phasereaction component of the invention, described above, carrying aplurality of hydroxylamine or protected hydroxylamine groups of formula(A) or (B).

In the process of the invention. the solid phase reaction component maybe in the form of a finely divided solid, or a web, membrane or openpore matrix. For example, the liquid reaction medium may be mixed withthe solid phase reaction product by passing the former through a liquidpermeable bed or column of the latter, or by contacting the former witha discrete quantity of the latter in finely divided form in a reactionvessel.

The solid phase reaction component and process of the present inventionare applicable in syntheses of the following general types, Schemes 1and 2: ##STR13##

In schemes 1 and 2, the vertical wavy line represents the base solidsubstrate, L represents a linker group, P₁ and P₂ are as defined above,and B₁, . . . B_(n) are the reaction building blocks for the synthesisof the desired product. In both cases, the desired product is releasedfrom the solid phase support by acid hydrolysis or photolysis of thebond designated (a) in formula (A) or (B), and the protecting group P₁or P₂ may be removed in the same or a separate step depending on itsidentity.

If desired, the intermediate solid phase reaction product can byisolated and physically partitioned into a plurality of portions afterthe addition of each building block B₁. . . B_(n). Each resultantportion may then be reacted with a different next building block. Thisprocess facilitates the parallel synthesis of a multiplicity ofdifferent hydroxamate end products.

Alternatively, the solid phase reaction component can be physicallypartitioned into a plurality of portions and each resultant portion maythen be reacted with a different first building block B₁. The separateportions may then be mixed to form a single portion which is then splitinto a plurality of portions. Each resultant portion may then be reactedwith a different second building block. The separate portions may thenbe mixed to form a single portion which is then split into a pluralityof portions for the coupling of the different second building blocks B₂.This process may be repeated a number of times until the final buildingblock B_(n) has been added. This process facilitates the combinatorialsynthesis of a mixture of a multiplicity of different hydroxamate endproducts.

Specific applications of the process of the invention are thepreparation of hydroxamic acids of the three classes referred to above,namely peptidyl, succinyl and arylsulphonamido hydroxamates.

For the synthesis of peptidyl hydroxamates (formula (I) above) by, forexample, Scheme 1, the building blocks B₁ . . . B_(n) represent theamino acids for sequential coupling. The first amino acid is coupled tothe groups A carried by solid phase reaction component of the invention,for example one of the preferred solid phase reaction components of theinvention carrying a plurality of groups of formula (IV) or (IVA). Inthe latter case, the amino group of the amino acid component may beprotected as a 9-fluorenylmethoxycarbonyl ("Fmoc") derivative and thecoupling of the amino acid carboxyl group to the nitrogen of the group Acarried by the solid phase reaction component of the invention may befacilitated by the use of a coupling agent such as is commonly used inpeptide synthesis, for example a carbodiimide (e.g.diisopropylcarbodiimide), a phosphonium salt (e.g.benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphoniumhexafluorophosphate) or a uronium salt (e.g.2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate), with the optional addition of an additive such as1-hydroxybenzotriazole. Alternatively, the coupling reaction may beconducted by preforming an active ester derivative (e.g.pentafluorophenyl ester) of the carboxylic acid component and optionallyconducting the reaction in the presence of an additive such as1-hydroxybenzotriazole. The Fmoc amino protecting group may then beremoved by treatment of the reaction product with a basic amine such aspiperidine. During the coupling reaction any groups in the amino acidwhich are potentially reactive under the coupling conditions may beprotected from such reaction as is usual in peptide synthesis. Additionof the second and any subsequent amino acids proceeds in a similarmanner until the desired peptidyl hydroxamate is finally cleaved fromthe solid substrate, eg by acid hydrolysis of the bond designated (a),and any protecting groups present in the molecule may be removed beforeduring or after such cleavage, depending on the nature of the protectinggroups in question.

For the synthesis of succinyl hydroxamates (formula (II) above) by, forexample, Scheme 1, an appropriate choice of building blocks B₁ . . .B_(n) to effect the synthesis is made. The first building block willgenerally be a carboxylic acid (also containing a site suitable forcoupling the next building block) which is coupled to the groups Acarried by solid phase reaction component of the invention, for exampleone of the preferred solid phase reaction components of the inventioncarrying a plurality of groups of formula (IV) or (IVA). Again thecoupling of the carboxyl group of the first building block to thenitrogen of the group A carried by the solid phase reaction component ofthe invention may be facilitated by the use of a coupling agent such asis commonly used in peptide synthesis, for example a carbodiimide (e.g.diisopropylcarbodiimide), a phosphonium salt (e.g.benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphoniumhexafluorophosphate) or a uronium salt (e.g.2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate), with the optional addition of an additive such as1-hydroxybenzotriazole. Alternatively, an activated ester, for example apentafluorophenyl ester, of the first carboxylic acid building block maybe coupled to the group A carried by the solid phase reaction componentof the invention in the presence of dimethylaminopyridine. A particularfirst carboxylic acid building block may be one of formula (IX)##STR14## wherein R₂ and R₃ are groups dictated by the desired succinylhydroxamate final product and R₁₄ is a carboxyl protecting group whichis compatible with solid phase synthesis such as an allyl group. Wherethe compound (IX) is coupled to the solid phase reaction components ofthe invention in this way, the R₁₄ carboxyl protecting group in theresultant product may be converted to a carboxylic acid group bydeprotection. In the case where R₁₄ is an allyl group deprotection maybe acheived by palladium catalysis. Suitable reagents for this step arePd(PPh₃)₄ or Pd(OAc)₂ /triphenylphosphine, and the reaction is usuallyconducted in the presence of an allyl acceptor such as morpholine. Thecarboxylic acid group thus generated may then be used as the point ofattachment for the second building block, which will generally be anamine of formula (X) ##STR15## wherein R₄ -R₆ are again groups dictatedby the desired succinyl hydroxamate final product. The methods describedabove and common in peptide synthesis are used to effect the coupling ofthe amine group of (X) with the carboxylic acid group of the firstbuilding block now carried on the solid phase reaction component. Duringthe coupling reactions of the foregoing assembly process, any groups inthe first and second building blocks which are potentially reactiveunder the coupling conditions may be protected from such reaction as isusual in peptide synthesis. After assembly of the desired succinylhydroxamate, the product may be cleaved from the solid substrate, eg byacid hydrolysis of the bond designated (a), and any protecting groupspresent in the molecule may be removed before, during or after suchcleavage, depending on the nature of the protecting groups in question.

For the synthesis of arylsulphonamide hydroxamates (formula (III) above)by the method of, for example, Scheme 1, an appropriate choice ofbuilding blocks B₁ . . . B_(n) to effect the synthesis is made. Thefirst building block will either be an alpha amino acid in which thealpha amino group is protected (for example as a9-fluorenylmethoxycarbonyl ("Fmoc") or allyloxycarbonyl ("Aloc")derivative), and the substituents on the alpha C atom (corresponding toR₇ and R₈ in formula (III)) are dictated by the desired arylsulphonamidehydroxamate final product or an alpha halo acid of formula (XI)##STR16## wherein R₇ and R₈ are dictated by the desired arylsulphonamidehydroxamate final product and Hal is a halogen such as bromo. This firstbuilding block (protected alpha amino acid or alpha halo acid) iscoupled to the groups A carried by solid phase reaction component of theinvention, for example one of the preferred solid phase reactioncomponents of the invention carrying a plurality of groups of formula(IV) or (IVA). In the latter case, the coupling of the protectedalpha-amino acid or alpha-halo acid carboxyl group to the nitrogen ofthe group A carried by the solid phase reaction component of theinvention may be facilitated by the use of a coupling agent such as iscommonly used in peptide synthesis, for example a carbodiimide (e.g.diisopropylcarbodiimide), a phosphonium salt (e.g.benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphoniumhexafluorophosphate) or a uronium salt (e.g.2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate), with the optional addition of an additive such as1-hydroxybenzotriazole. Alternatively this first building block may becoupled to the group A carried by the solid phase reaction component ofthe invention as an activated ester, for example a pentafluorophenylester, in the presence of dimethylaminopyridine.

In the case where the first building block is a protected alpha-aminoacid the N-protecting group (eg Fmoc or Aloc) may be removed bytreatment of the reaction product with a basic amine such as piperidinein the case of base labile protecting groups (e.g. Fmoc) or treatmentwith a suitable palladium catalyst in the case of palladium labileprotecting groups (e.g. Aloc). The free amino group then serves as thesite for formation of the appropriate arylsulphonamide and alkylation ofthe sulphonamide nitrogen to complete the synthesis of the desiredarylsulphonamide hydroxamate, which can then be cleaved from the solidsubstrate, eg by acid hydrolysis of the bond designated (a).

In the case where the first building block is an alpha-halo acid thehalogen may be displaced by reaction with an amine to form a secondaryamine which serves as the site for formation of the sulphonamide tocomplete the synthesis of the desired arylsulphonamide hydroxamate,which can then be cleaved from the solid substrate, eg by acidhydrolysis of the bond designated (a).

Examples 1-5 disclose the preparation of solid phase reaction componentsof the invention and Examples A-H illustrate their utility in thepreparation of biologically active hydroxamic acid derivatives.

The amino acids used in the examples below were commercially availableor were prepared according to literature procedures.

The following abbreviations have been used throughout:

BPB Bromophenol blue

DMF N,N-Dimethylformamide

DMSO Dimethylsulfoxide

Fmoc 9-Fluorenylmethoxycarbonyl

pyBOP Benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate

HOBt N-Hydroxybenzotriazole

THF Tetrahydrofuran

TFA Trifluoroacetic acid

TLC Thin layer chromatography

TBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate

Pmc 2,2,5,7,8-Pentamethylchroman-6-sulfonyl

Z Benzyloxycarbonyl

¹ H and ¹³ C NMR spectra were recorded using either a Bruker AC 250Espectrometer at 250.1 and 62.9 MHz respectively, or on a Bruker AMX500at 500.13 and 125.7 MHz respectively. Elemental microanalyses wereperformed by Medac Ltd (Department of Chemistry, Brunel University,Uxbridge, Middlesex UB8 3PH).

EXAMPLE 1

4-(O-Methylhydroxylamine)phenoxymethyl-copoly(styrene-1%divinylbenzene)-resin (100-200 mesh) ##STR17##

4-(Hydroxymethyl)phenoxymethyl-copoly(styrene-1% divinylbenzene)-resin(100-200 mesh) ("Wang" resin) (1.83 g, 0.7 mmol/g loading, 1.28 mmol)was suspended in dry THF (20 cm³) and gently agitated for 30 minutesunder a blanket of argon. N-Hydroxyphthalimide (624 mg, 3.82 mmol) andtriphenylphosphine (1.005 g, 3.82 mmol) were added and the mixture wasagitated until these reagents dissolved. Diethylazodicarboxylate (721mg, 3.28 mmol) was added by cannula to give a bright red solution andthe mixture was shaken. Most of the colour dissipated from the reactionmixture after 45 minutes. After 18 h the resin was collected byfiltration, washed successively with THF, DMF, dichloromethane, methanoland finally thoroughly with dichloromethane and then dried in vacuo;v_(max) (KBr) 01726 (vs), 1687, 1593 cm⁻¹.

The resin from above was suspended in DMF (10 cm³) and hydrazine(˜100%)(1.5 cm³) was added. The pale yellow solution was heated at 50°C. for 2 h and then gently agitated overnight. The desired resin(4-(O-methylhydroxylamine)phenoxymethyl-copoly(styrene-1%divinylbenzene)-resin (100-200 mesh)) was filtered, and washed with DMF(2×15 cm³), CH₂ Cl₂ (2×15 cm³), methanol (20 cm³) and finally with CH₂C₂ (3×15 cm³) and then dried. Ir (KBr) showed the absence of anycarbonyl containing substituent confirming hydrazinolysis of thephthalimide. Elemental analysis of the resin confirmed the presence ofnitrogen and suggested a loading of 0.68 mmol/g: C 89.17, H 7.70, N0.95%. [Note: The loading obtained varied for different batches of theresin due to variability in the loading of the starting Wang resin, butin each case elemental analysis suggested that quantative conversion wasachieved.]

EXAMPLE 2

O-Hydroxylamine-2'-chlorotrityl-copoly(styrene-1%-divinylbenzene)-resin(200-400 mesh) ##STR18##

Chloro-2'-chlorotrityl-copoly(styrene-1%-divinylbenzene) resin (200-400mesh)(0.5 g, 1.5 mmol/g, 0.75 mmol) was allowed to swell in dry DMF (3cm³) for 10 minutes. Diisopropylamine (0.398 cm³, 2.28 mmol) was addedto a solution of N-hydroxyphthalimide (0.367 g, 2.25 mmol) in DMF (3cm³) and the subsequent bright red solution added in one portion to theresin suspension and the resulting mixture was then gently agitated for4 hr under an argon atmosphere. The resin was collected by filtrationand washed thoroughly with DMF (3×10 cm³), dichloromethane (2×10 cm³),followed by methanol (3×10 cm³, 3 cycles), dichloromethane (2×10 cm³)and dried. The pale yellow resin had u_(max) (KBr) 1737 (vs) cm⁻¹.

THF (5 cm³) was added to the resin which was allowed to swell for 15minutes. Hydrazine hydrate (4 cm³) was added to the mixture and theresulting mixture was gently agitated for 3 hr. Following filtration andwashing as above the resin was dried. It exhibited no stretch at 1737cm⁻¹ in the i.r spectrum and was stained blue by a 2% solution ofbromophenol blue (BPB) in DMF.

EXAMPLE 3

4-[4-(O-Methylhydroxylamine)-3-methoxyphenoxy]-(N-4-methylbenzhydryl)butyramide-copoly(styrene-1%-divinylbenzene)-resin(100-200 mesh) ##STR19##

Following the analogous procedure to that described in Example 1,4-[4-hydroxymethyl-3-methoxyphenoxy]-(N-4-methylbenzhydryl)butyramidecopoly(styrene-1%-divinylbenzene)-resin(100-200 mesh) (0.87 mmol/g loading) was treated with 3 equivalents oftriphenylphosphine, N-hydroxyphthalimide and diethylazodicarboxylate inTHF to give the N-hydroxyphthalimido derivatised resin which ontreatment with hydrazine hydrate gave the desired4-[4-(O-methylhydroxylamine)-3-methoxyphenoxy]-(N-4-methylbenzhydryl)-butyramidecopoly(styrene-1%-divinylbenzene)-resin.

EXAMPLE 4

4-(2',4'-Dimethoxyphenyl-O-methylhydroxylamine)-phenoxymethyl-copoly(styrene-1%-divinylbenzene)-resin(100-200 mesh) ##STR20##

Following the analogous procedure to that described in Example 1,4-(2',4'-dimethoxyphenyl-O-methylhydroxy)-phenoxymethyl-copoly(styrene-1%divinylbenzene)-resin (100-200 mesh) (0.54 mmol/g loading) was treatedwith 3 equivalents of triphenylphosphine, N-hydroxyphthalimide anddiethylazodicarboxylate in THF to give the N-hydroxyphthalimidoderivatised resin which on treatment with hydrazine hydrate gave thedesired4-(2',4'-dimethoxyphenyl-O-methylhydroxylamine)-phenoxymethyl-copoly(styrene-1%-divinylbenzene)-resin.

EXAMPLE 5

4-[4-(1-Aminooxyethyl)-2-methoxy-5-nitrophenoxy]-(N-4-methylbenzhydryl)butyramide-copoly(styrene-1%-divinylbenzene)-resin(100-200 mesh) ##STR21##

Methyl 4-(4-acetyl-2-methoxyphenoxy)butyrate (10 g) was added to cold70% nitric acid (140 cm³) and the resulting orange solution was stirredfor 2 h and then poured into a beaker containg ice-water (1 dm³). Theslurry warmed with stirring overnight to give a yellow solid which wascollected and recrystallised from methanol to give methyl4-(4-acetyl-2-methoxy-5-nitrophenoxy)butyrate as a pale yellow solid(8.5 g). ¹ H NMR (CDCl₃) δ2.21 (2 H, m, J=6.6 Hz, CCH₂ C), 2.50 (3 H, s,MeCO), 2.57 (2 H, t, J=7.1 Hz, CH₂ CO), 3.7 (3 H, s, OMe), 3.99 (3 H, s,OMe), 4.16 (2 H, t, J=6.2 Hz, CH₂ O), 6.75 (1 H, s, aromatic) and 7.62(1 H, s, aromatic).

Methyl 4-(4-acetyl-2-methoxy-5-nitrophenoxy)butyrate (5.06 g) inmethanol (300 cm³) was cooled with stirring at 0° C. under argon. Sodiumborohydride (640 mg) was added portionwise and the resulting solutionstirred at 40° C. overnight. Further sodium borohydride (500 mg) wasadded as TLC analysis indicated that starting material remained. After afurther 3 h TLC analysis indicated that the reaction was complete. Thereaction was cooled, acidified with dilute (1 M) hydrochloric acid andevaporated to dryness. The residue was dissolved in ethyl acetate (250cm³) washed with dilute aqueous acid and brine, dried over magnesiumsulphate and filtered. The solvent was evaporated to leave a yellow oilwhich solidified on standing. After recrystallisation frommethanol/diethyl ether, methyl4-[4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy]butyrate was obtained asyellow needles (5.04 g, ˜100%). ¹ H NMR (CDCl₃) δ1.52 (3 H, d, J=8.3 Hz,MeCHO), 2.18 (2 H, m, CCH₂ C), 2.53 (2 H, t, J=6.5 Hz. CH₂ CO), 3.71 (3H, s, OMe), 3.95 (3 H, s, OMe), 4.13 (2 H, t, J=7.3 Hz, CH₂ O), 5.52 (1H, q, J=8.2 Hz, CHOH), 7.29 (1 H, s, aromatic) and 7.57 (1 H, s,aromatic).

Methyl 4-[4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy]butyrate (5.04 g)from above was stirred with lithium hydroxide monohydrate in methanolusing conventional protocols.4-[4-(1-Hydroxyethyl)-2-methoxy-5-nitrophenoxy]butyric acid was obtainedas an amorphous yellow solid (4.0 g, 83%). ¹ H NMR (CDCl₃) δ1.34 (3 H,d, J=11 Hz, MeCOH), 2.03 (2 H, m, CCH₂ C), 2.38 (2 H, t, J=7.3 Hz, CH₂CO), 3.81 (3 H, s, OMe), 3.96 (2 H, t, J=7.4 Hz, CH₂ O), 4.13 (2 H, brs,OH), 5.38 (1 H, q, J=10.8 Hz, CHOH), 7.19 (1 H, s, aromatic, and 7.43 (1H, s, aromatic).

4-Methylbenzhydrylamine-copoly(styrene-1%-divinylbenzene)-resin (100-200mesh) (2.0 g, 0.7 mmol/g loading, 1.4 mmol) was placed in DMF (10 cm³).4-[4-(1-Hydoxyethyl)-2-methoxy-5-nitrophenoxy]butyric acid (1.51 g, 4.8mmol), HOBt (734 mg, 4.8 mmol) and TBTU (1.54 g, 4.8 mmol) were addedand the mixture agitated. N-Methylmorpholine (0.88 cm³, 8 mmol) wasadded and the resulting mixture was gently agitated for 18 h in a flaskprotected from light by aluminium foil. The bright yellow resin (2.45 g)was collected by filtration, thoroughly washed as described in Example 1and dried in vacuo. The Kaiser test was negative which contrasted withthat of the unsubstituted resin. u_(max) (KBr) 1672 (s) cm⁻¹.

The above resin (2.41 g) was placed in a small conical flask under argonand THF (25 cm³) was added. The resin was allowed to swell for 20minutes and triphenylphosphine (1.67 g, 6.36 mmol) andN-hydroxyphthalimide (1.038 g, 6.36 mmol) were added. The mixture wasshaken together in the dark to dissolve the reagents and then cooled to0°. Diethylazodicarboxylate (1.0 cm³, 6.36 mmol) was added and thesolution turned red. After shaking the reaction at room temperature for48 h the resin was then collected by vacuum filtration, washed asdescribed in Example 1 and dried in vacuo. u_(max) (KBr) 1792, 1734 (vs)and 1669 (s) cm⁻¹.

Hydrazine (2.2 cm³) was added to a suspension of this resin (2.7 g) inDMF (15 cm³) under argon. The mixture was shaken at room temperature inthe dark overnight and the resin(4-[4-(1-aminooxyethyl)-2-methoxy-5-nitrophenoxy]-(N-4-methylbenzhydryl)-butyramide-copoly(styrene-1%-divinylbenzene)-resin(100-200 mesh)) was then collected, washed and dried as described inExample 1. It showed no phthalimide carbonyl stretch in the IR spectrum.u_(max) (KBr) 1673 (s) cm⁻¹.

Example A

Use of the resin from Example 1 in the preparation of a peptidehydroxamate derivative:Benzyloxycarbonyl-L-prolyl-L-leucyl-L-alanylhydroxamic acid ##STR22##

The resin (4-(O-methylhydroxylamine)phenoxymethyl-copoly(styrene-1%divinylbenzene)-resin (100-200 mesh)) as prepared in Example 1 (200 mg,0.14 mmol) was suspended in DMF (6 cm³). Fmoc-Alanine (217 mg, 0.7mmol), HOBt (107 mg, 0.7 mmol), pyBOP (364 mg. 0.7 mmol) and N-methylmorpholine (0.123 cm³, 1.12 mmol) were added and the suspension wasagitated for 4 h. The resin was filtered, washed with DMF and thensuspended and agitated in a 20% solution of piperidine in DMF for 40minutes. The solution was drained from the resin which was washed withDMF, dichloromethane, methanol, dichloromethane and finally resuspendedin DMF (6 cm³). Following the same protocols described aboveFmoc-leucine and benzyloxycarbonylproline were then successively coupledto the resin.

On completion the resin was washed thoroughly and dried in vacuoovernight. The resin was treated with 95% aqueous TFA (10 cm³) for 75minutes. The TFA solution was collected by filtration, the resin washedwith more TFA (˜2 cm³) and the combined filtrates evaporated to leave agum which was triturated with ether to give a white solid. TLC of thewhite solid indicated a single compound which gave a positive ferricchloride test indicative of a hydroxamic acid. The solid was collectedand dried to give the desiredbenzoxycarbonyl-L-prolyl-L-leucyl-L-alanyl-hydroxamic acid (54 mg,˜90%); ¹ H NMR (CDCl₃) δ7.35 (5 H, m, aromatic), 5.15 (1 H, J=7 Hz,CH_(a) H_(b) O), 5.13 (1 H, d, J=7 Hz, CH_(a) H_(b) O), 4.48 (1 H, m),4.33 (1 H, m), 4.23 (1 H, m), 3.52 (2 H, m, pro), 1.9-2.2 (3 H, m, pro),1.48-1.78 (2 H, m), 1.48 (3 H, d, J=8.2 Hz, ala), 1.25 (2 H, m, CH₂leu), 0.86 (6 H, m, CH₃ -leu); ¹³ C NMR (CDCl₃) δ173.5, 172.2, 169.3,156.4, 136.0. 128.6, 128.4, 127.9, 67.8, 61.5, 53.2, 47.4, 47.3, 39.7,29.6, 25.1, 24.7, 22.6, 21.4, 17.0.

Example B

Use of the resin from Example 1 in the preparation of a library of `lefthand side` inhibitors of matrix metalloproteinases

    Z--AA.sub.3 --AA.sub.2 --AA.sub.1 --NHOH

1 g of the resin(4-(O-methylhydroxylamine)phenoxymethyl-copoly(styrene-1%divinylbenzene)-resin (100-200 mesh)) of Example 1 was placed in each of10 sealable polypropylene tubes (BioRad) and swelled in DMF (1 cm³). 2.5mmol (4 equivalents) of one of the Fmoc-amino acids, alanine, D-alanine,phenylalanine, isoleucine, leucine, D-leucine, norleucine, methionine,valine, norvaline, was placed in a tube followed by HOBt (3.85 cm³ of a0.65 M solution in DMF). Diisopropylcarbodiimide (0.4 cm³) was added toeach tube which were then sealed and gently agitated for 18 h when theywere drained of solvent and washed thoroughly with DMF. A solution of30% piperidine in DMF (6 cm³) was added to each tube, the resin wasmixed by shaking and then allowed to stand in the solution for 40minutes, the solvent was drained and the resin washed successively withDMF, methanol and dichloromethane as described above in Example 1 anddried.

The resin from each individual tube was then portioned into 10 tubes(˜100 mg/tube) and a 1% solution of bromophenol blue in DMF (1 cm³)added to swell and stain the resin.

0.25 mmol of one of the Fmoc-amino acids alanine, D-alanine, arginine(N^(G) -Pmc), aspartic acid-b-tert-butyl ester, leucine, proline,norleucine, glutamine-γ-trityl, thioproline, ε-Boc-lysine was added toone of the tubes derived from the sub-division of the resin from theoriginal ten tubes. HOBt (0.38 cm³ of a 0.65 M solution in DMF) wasadded to every tube followed by diisopropylcarbodiimide (39 mm³). Thetubes were then sealed and agitated overnight. At this time the colourhad discharged from almost every tube. A further 0.05 mmol portion eachof the Fmoc-amino acid and diisopropylcarbodiimide was added to thosetubes which still showed a blue-green colouring; this was dischargedwithin 30 minutes of the addition of the extra reagent. The resin fromthese tubes was then pooled back into the tube from where it hadoriginated, washed with DMF and then treated with 30% piperidine in DMFfor 40 minutes and washed thoroughly as above.

The resin from each of these ten tubes was then redivided into 5 tubes(˜200 mg/tube) and allowed to swell in a 1% solution of bromophenol bluein DMF (1 cm³). 5 mmol of one of the Z-amino acids, alanine,phenylalanine, proline, leucine, aspartic acid-β-tert-butyl ester, wasadded to one tube from each sub-division followed by HOBt (5 cm³ of a1.0 M solution in DMF) and by diisopropylcarbodiimide (0.78 cm³). Thetubes were gently agitated for 5 h by when the blue colour had beendischarged in all tubes. The resin was then repooled into the originaltubes as above and thoroughly washed with DMF, dichloromethane andmethanol as described previously. The resin, maintained in the 10 tubes,was then dried in vacuo overnight.

5 cm³ of a solution of 5% phenol, 5% water, 2% triisopropylsilane in TFAwas added to the resin in each tube and periodically shaken over 60minutes. The TFA solution was collected by filtration and the resinwashed with a further 2 cm³ of TFA. The washings and filtrate from eachindividual tube were combined and the solvent evaporated to leave aresidue which was triturated with cold diisopropylether/hexane to givewhite solid precipitates. After removal of most of the organic solventby decanting the precipitates were then lyophilised from 50% aqueousacetontrile to give the desired 10 mixtures of 50 tripeptide hydroxamicacids.

Each of the ten mixtures of peptidyl hydroxamic acids was assayed foractivity as inhibitors of metalloproteinase enzymes. Those pools showingthe greatest inhibitory activity were then subjected to an iterativedeconvolution procedure to identify individual active inhibitors.

Example C

Use of the resin from Example 1 in the preparation of a sulfonamidehydroxamate derivative:2-(N-Decyl-2-acetamido-4-methylthiazol-5-ylsulfonamido)acetohydroxamicacid ##STR23##

Bromoacetic acid (167 mg, 1.2 mmol) and diisopropylcarbodiimide (94 mm³,0.6 mmol) were added to a suspension of the resin(4-(O-methylhydroxylamine)phenoxymethyl-copoly(styrene-1%divinylbenzene)-resin (100-200 mesh)) prepared according to Example 1(250 mg, 0.1 mmol) in DMF (6 cm³). The mixture was agitated for 90minutes and then the solution was drained from the resin which waswashed and dried as described above in Example 1. The resin was soakedin DMSO (2 cm³) and a solution of n-decylamine in DMSO (5 cm⁵ of a 2 Msolution. 10 mmol) was added and the mixture shaken for 4 h before theresin was drained and washed thoroughly as before. The resin was thensuspended in DMF and 2-acetamido-4-methyl-5-thiazolesulfonylchloride(102 mg, 0.4 mmol) was added. The mixture was heated at 6° for 8 h andthen the resin was drained, washed and dried as described previously.Cleavage from the resin as in Example A gave a white solid which waspurified by column chromatography (10% methanol--dichloromethane;silica) to give the desired2-(N-decyl-2-acetamido-4-methylthiazol-5-ylsulfonamido)-acetohydroxamicacid (23 mg, 51%) as a white solid; ¹ H NMR (CDCl₃) δ3.83 (2 H, m), 3.05(2 H, m), 2.52 (3 H, s, Me), 2.35 (3 H, s, NHCOMe), 1.69 (2 H, m),1.15-1.25 (14 H, m, alkyl), 0.87 (3 H, t, J=7.2 Hz, CH₃ -alkyl).

Example D

Use of the resin from Example 1 in the preparation of an array ofsulfonamide hydroxamate inhibitors of matrix metalloproteinases##STR24##

Sulfonamide hydroxamic acid derivatives were prepared as a combinatorialarray of individual compounds in purity sufficient for fast throughputassays. The resin(4-(O-methylhydroxylamine)phenoxymethyl-copoly(styrene-1%divinylbenzene)-resin (100-200 mesh)) was loaded with brornoacetic acidas described in Example C. Elemental analysis of this different batch ofresin confirmed that coupling had taken place: C 83.34, H 7.04, N 0.86,Br 5.40% and suggested a loading of 0.65 mmol. 1 g of this resin wasplaced in each of 36 plastic Econopac tubes (BioRad) and swelled in DMF(5 cm³). The excess DMF was drained off and the tubes grouped into ninegroups of four tubes. A solut ion (5 cm³ of a 2 M solution in either DMFor DMSO) of one of the amines, benzylamine, 4-methylbenzylamine,4-methoxybenzylamine, 4-fluorobenzyiamine, 4-chlorobenzylamine,pyridin-3-ylmethylamine, thien-2-ylmethylamine, furan-2-ylmethylamineand 3,4-dioxymethylenebenzylamine was each added to each of four tubes(within one of the nine groups) which were then sealed and mixedthoroughly for 2 h. The solvent was removed, the resin washed as inExample C and reswelled in DMF. The tubes were regrouped into fourgroups each of nine tubes. Following a protocol analogous to that usedin Example C, a DMF solution of 10 equivalents of one of the sulfonylchlorides, 3-chloropropylsulfonyl chloride, hexylsulfonyl chloride,octylsulfonyl chloride and decylsulfonyl chloride was added to each ofthe nine tubes (within one of the four groups) so that all 36combinations of sulfonamides were prepared. After removal of the soventby filtration, thorough washing with DMF, methanol and dichloromethaneas above, and drying under vacuum for several hours the products werecleaved from the resin as in Example C directly into plastic centrifugetubes. The TFA was removed by centrifugal evaporation and the resultinghydroxamic acids lyophilised from aqueous acetonitrile. All samples gavepositive ferric chloride spots on TLC analysis {10% methanol inDCM/silica} (Table). All 36 samples were submitted for assay asinhibitors of metalloproteinases from which active compounds wereidentified.

                  TABLE                                                           ______________________________________                                        TLC {10% methanol in DCM/silica} R.sub.f values for the compounds              of Example D                                                                            Rb                                                                 Ra         --(CH.sub.2).sub.3 Cl                                                                   --C.sub.6 H.sub.13                                                                     --C.sub.8 H.sub.17                                                                    --C.sub.10 H.sub.21                     ______________________________________                                        C.sub.6 H.sub.5 --                                                                       0.25      0.2      0.45    0.45                                      4-MeC.sub.6 H.sub.4 -- 0.35 0.4 0.4 0.4                                       4-MeOC.sub.6 H.sub.4 -- 0.5 0.35 0.5 0.4                                      4-FC.sub.6 H.sub.4 -- 0.3 0.3 0.5 0.5                                         4-ClC.sub.6 H.sub.4 -- 0.15 0.2 0.4 0.4                                       pyridin-3-yl 0.0 0.05 0.0 0.05                                                thien-2-yl 0.1 0.35 0.65 0.6                                                  furan-2-yl 0.15 0.3 0.3 0.4                                                   3,4-(CH.sub.2)O.sub.2 C.sub.6 H.sub.3 -- 0.3 0.4 0.5 0.45                   ______________________________________                                    

Example E

Use of the resin from Example 2 in the preparation of a peptidehydroxamate derivative:Benzyloxycarbonyl-L-phenylalanyl-L-alanylhydroxamic acid ##STR25##

FMOC-Alanine (187 mg, 0.6 mmol), HOBt (92 mg, 0.6 mmol) and TBTU (194mg, 0.6 mmol) were dissolved together in DMF (5 cm³).Diisopropylethylamine (0.183 cm³, 1.05 mmol) was added and the resultingsolution added to pre-swelled (in DMF) modified resin from Example 2(O-hydroxylamine-2'-chlorotrityl-copoly(styrene-1%-divinylbenzene)-resin(200-400 mesh)) (100 mg, 0.15 mmol) and the mixture was then shakengently together for 12 h. The resulting resin was then filtered, washedthoroughly with DMF and treated with 20% piperidine in DMF (7 cm³) for30 minutes. After removal of the solvent and thorough washing asdescribed in Example 1 the resin was resuspended in DMF (1 cm³). Asolution of Z-phenylalanine (180 mg, 0.6 mmol), HOBt (92 mg, 0.6 mmol)and diisopropylcarbodiimide (94 mm³, 0.6 mmol) in DMF (5 cm³) was addedand the resin suspension was gently agitated overnight by which time theinitially blue staining of BPB had been discharged. The resin was thendrained and washed as described in Example 1 and dried in vacuo. It hadu_(max) (KBr) 1668 (s) cm⁻¹.

The resin from above was suspended in a 20% solution of TFA indichloromethane, containing 1% (v/v) of triethylsilane, under argon (5cm³) for 45 minutes. The solution was removed from the resin byfiltration, the resin washed with a further portion (1 cm³) of thecleavage mixture, the filtrate and washings combined and evaporated toleave benzyloxycarbonyl-L-phenylalanyl-L-alanylhydroxamic acid as awhite solid. TLC analysis indicated a single compound, R_(f) 0.45 (10%methanol in dichloromethane) which gave a positive ferric chloride test.¹ H NMR (CDCl₃) δ1.36 (3 H, d, J=9 Hz, Me alanine), 3.12 (2 H, m, CH₂Ph), 4.14 (1 H, m, CH), 4.4 (1 H, m, CH), 5.13 (2 H, brs, OCH₂ Ph),7.71-7.33 (˜10 H, m, aromatic) 7.8 (1 H, d, NH).

Example F

Use of the resin from Example 3 in the preparation of a sulfonamidehydroxamate derivative: 2-(N-Benzyl-benzylsulfonamido)-acetohydroxamicacid ##STR26##

The resin of Example 3(4-[4-(O-methylhydroxylamine)-3-methoxyphenoxy]-(N-4-methylbenzhydryl)butyramide-copoly(styrene-1%-divinylbenzene)-resin(100-200 mesh)) (0.5 g) was suspended in DMF (3 cm³).Diisopropylcarbodiimide (0.3 cm³) was added to a solution of bromoaceticacid (670 mg) in DMF (3 cm³) and after 3 minutes the resulting solutionwas added to the resin suspension and gently mixed together for 2 h. Theresin was collected by filtration and washed thoroughly with DMF,methanol, methanol-dichloromethane, dichloromethane and ether. It wasthen dried in vacuo at 45° C. for 18 h. Elemental analysis of the resinsuggested a loading of 0.66 mmol/g: C; 79.11; H, 6.78; N, 2.08; Br,5.25%.

The resin from above swelled in DMF (3 ml) was treated with (0.33 cm³, 3mmol) benzylamine and gently agitated at room temperature overnight. Thesolvent was removed by filtration and the resin washed as describedabove in Example 1 and dried. The resin was swelled in DMF (3 ml), andphenylmethylsulfonyl chloride (0.8 g, 4 mmol) was added and the mixturewas gently agitated for 2 h at room temperature. The resin was filteredand washed as described above in Example 1 and dried. A solution of 1%TFA in DCM (3 ml) was added to the resin at room temperature and themixture gently agitated for 1 h. The resin was filtered and washed withDCM, methanol and DCM, the filtrates combined and evaporated underreduced pressure to give crude2-(N-benzyl-benzylsulfonamido)-acetohydroxamic acid (0.39 g): TLC ferricchloride positive spot R_(f) 0.15 {10% methanol in DCM/silica}.

Example G

Use of the resin from Example 4 in the preparation of a sulfonamidehydroxamate derivative: 2-(N-Benzyl-benzylsulfonamido)-acetohydroxamicacid

This resin of Example 4(4-(2',4'-dimethoxyphenyl-O-methylhydroxylamine)phenoxymethyl-copoly(styrene-1%-divinylbenzene)-resin(100-200 mesh)) (0.25 g) was suspended in DMF (3 cm³).Diisopropylcarbodiimide (0.24 cm³) was added to a solution ofbromoacetic acid (420 mg) in DMF (3 cm³) and after 3 minutes theresulting solution was added to the resin suspension and gently mixedtogether for 2 h. The resin was collected by filtration and washedthoroughly with DMF, methanol, methanol-dichloromethane, dichloromethaneand ether. It was then dried in vacuo at 45° C. for 18 h. Elementalanalysis of the resin suggested a loading of 0.58 mmol/g: C; 84.31; H,7.28; N, 0.82; Br, 4.65%.

Following the procedure of Example F the above resin was treated in turnwith benzylamine and phenylmethylsulfonyl chloride. A solution of 10%acetic acid in DCM (3 ml) was added to the resin at room temperature andthe mixture gently agitated for 2 h. The resin was filtered and washedwith DCM. methanol and DCM, the filtrates combined and evaporated underreduced pressure to give crude2-(N-benzyl-benzylsulfonamido)-acetohydroxamic acid (0.22 g).

Example H

Use of the resin from Example 5 in the preparation of a peptidehydroxamate derivative:Benzyloxycarbonyl-L-prolyl-L-leucyl-L-alanylhydroxamic acid

Using procedures analogous to those described in Example A, thetripeptide Z-L-prolyl-L-leucyl-L-alanyl was synthesised attached to theresin of Example 5(4-[4-(1-aminooxyethyl)-2-methoxy-5-nitrophenoxy]-(N-4-methylbenzhydryl)-butyramide-copoly(styrene-1%-divinylbenzene)-resin(100-200 mesh)). Following drying, the elaborated resin (50 mg) inacetonitrile (1.5 cm³) was blanketed with argon in a stoppered reactionvial. The vial was placed 5 cm away from a 365 nm light source andirradiated for 15 h. TLC of the resulting solution indicated a ferricchloride positive component with R_(F) equivalent to that of thetripepeptide hydroxamic acid of Example A. HPLC of the solutionidentified the desired tripeptide as the major product of the reactionalong with an unidentified non-hydroxamic acid component.

What is claimed is:
 1. A solid phase reaction product comprising a solidsubstrate that is substantially insoluble in aqueous or organic reactionmedia, wherein the solid substrate comprises a base substrate carrying aplurality of covalently bound hydroxylamine or protected hydroxylaminegroups of formula (B): ##STR27## wherein P₂ is hydrogen or a hydroxylprotecting group and the bond designated (a) is (i) cleavable under acidconditions or by photolysis and (ii) covalently links the group (B) tothe base substrate via a linker group of formula (D): ##STR28## whereinthe base substrate is directly linked to the linker group of formula (D)via a R_(13A), and the hydroxylamine group of formula (B) is directlylinked to the linker group of formula (D) at *,wherein in formula(D):R₁₁ and R_(11A) independently represent hydrogen, C₁ -C₆ alkyl, orphenyl optionally substituted by one or more substituents selected fromC₁ -C₆ alkyl, C₁ -C₆ alkoxy, halogen, nitrile or NO₂ ; R₁₂ and R_(12A)independently represent hydrogen, C₁ -C₆ alkyl, C₁ -C₆ alkoxy, halogen,nitrile or NO₂ ; R_(13A) represents a bond or a group --(X¹)_(q) --Y--,wherein q is 0 or 1; X¹ represents --C(═O)--, --CH₂ --, --CH₂ C(═O)--,--O(CH₂)_(n) C(═O)--, --O(CH₂)_(n) C(═O)--(A¹)_(m) --, or --O(CH₂)_(n)C(═O)--(A¹)_(m) --B¹ --, wherein n is an integer from 1 to 6; m is 0 or1; A¹ represents --OCH(R¹)--NH--, wherein R¹ is the side chain of anatural or unnatural alpha amino acid, B¹ represents a spacer group--NH(CH₂)_(p) --, wherein p is 0 or an integer from 1 to 6; and Yrepresents --O-- or --NH--.
 2. The solid phase reaction product asclaimed in claim 1, wherein P₂ represents hydrogen.
 3. The solid phasereaction product as claimed in claim 1, wherein P₂ represents a hydroxylprotecting group.
 4. The solid phase reaction product as claimed inclaim 2, wherein R₁₁ and R_(11A) are each independently hydrogen,methyl, phenyl, 4-methylphenyl, 4-methoxyphenyl or 2,4-dimethoxyphenyl.5. The solid phase reaction product as claimed in claim 3, wherein R₁₁and R_(11A) are each independently hydrogen, methyl, phenyl,4-methylphenyl, 4-methoxyphenyl or 2,4-dimethoxyphenyl.
 6. The solidphase reaction product as claimed in claim 2, wherein R₁₂ and R_(12A)are each independently hydrogen or methoxy.
 7. The solid phase reactionproduct as claimed in claim 3, wherein R₁₂ and R_(12A) are eachindependently hydrogen or methoxy.
 8. The solid phase reaction productas claimed in claim 1, wherein P₂ represents hydrogen; R₁₁ and R_(11A)are each independently hydrogen, methyl, phenyl, 4-methylphenyl,4-methoxyphenyl or 2,4-dimethoxyphenyl; R₁₂ and R_(12A) are eachindependently hydrogen or methoxy; and R_(13A) represents a bond or agroup --(X¹)_(q) --Y--, wherein q is 0 or 1; X¹ represents --C(═O)--,--CH₂ --, --CH₂ C(═O)--, --O(CH₂)_(n) C(═O)--, --O(CH₂)_(n)C(═O)--(A¹)_(m) -- or --O(CH₂)_(n) C(═O)--(A¹)_(m) --B¹ --, wherein n isan integer from 1 to 6; m is 0 or 1; A¹ represents --OCH(R¹)--NH--,wherein R¹ is the side chain of a natural alpha amino acid; B¹represents a spacer group --NH(CH₂)_(p) --, wherein p is 0 or an integerfrom 1 to 6; and Y represents --O-- or --NH--.
 9. The solid phasereaction product as claimed in claim 1, wherein R_(13A) together withthe base substrate to which it is attached isoxymethyl-copoly(styrene-1%-divinylbenzene) resin,oxymethyl-copoly(styrene-2%-divinylbenzene)resin,oxyacetomidomethylpolyethyleneglycol-copoly(styrene-1%-divinylbenezene)resinoroxyacetomidomethylpolyethyleneglycol-copoly(styrene-2%-divinylbenzene)resin.10. The solid phase reaction product as claimed in claim 8, whereinR_(13A) together with the base substrate to which it is attached isoxymethyl-copoly(styrene-1%-divinylbenzene)resin,oxymethyl-copoly(styrene-2%-divinylbenzene)resin,oxyacetomidomethylpolyethyleneglycol-copoly(styrene-1%-divinylbenezene)resinoroxyacetomidomethylpolyethyleneglycol-copoly(styrene-2%-divinylbenzene)resin.